1,1′-bis(anilino)-4-,4′-bis(naphtalene)-8,8′-disulfonate acts as an inhibitor of lipoprotein lipase and competes for binding with apolipoprotein CII

Abstract

Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII), a component of plasma lipoproteins, for function in vivo. The hydrophobic fluorescent probe 1,1′-bis(anilino)-4,4′-bis(naphthalene)-8,8′ -disulfonate (bis-ANS) was found to be a potent inhibitor of LPL. ApoCII prevented the inhibition by bis-ANS, and was also able to restore the activity of inhibited LPL in a competitive manner, but only with triacylglycerols with acyl chains longer than three carbons. Studies of fluorescence and surface plasmon resonance indicated that LPL has an exposed hydrophobic site for binding of bis-ANS. The high affinity interaction was characterized by an equilibrium constant Kd of 0.10-0.26 μM and by a relatively high on rate constant kass = 2.0 × 104 M-1 S-1 and a slow off-rate with a dissociation rate constant k diss = 1.2 × 10-4 s-1. The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII.